The Streptococci make up a medically important genera of microbes known to cause several types of disease in humans, including, for example, otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid. Since its isolation more than 100 years ago, Streptococcus pneumoniae has been one of the more intensively studied microbes. For example, much of our early understanding that DNA is, in fact, the genetic material was predicated on the work of Griffith and of Avery, Macleod and McCarty using this microbe. Despite the vast amount of research with S. pneumoniae, many questions concerning the virulence of this microbe remain. It is particularly preferred to employ Streptococcal genes and gene products as targets for the development of antibiotics.
Infections caused by or related to Streptococcus pneumoniae are a major cause of human illness worldwide, and the frequency of resistance to standard antibiotics has risen dramatically over the last decade. Hence, there exists an unmet medical need and demand for new anti-microbial agents, vaccines, drug screening methods, and diagnostic tests for this organism.
Moreover, the drug discovery process is currently undergoing a fundamental revolution as it embraces “functional genomics,” that is, high throughput genome- or gene-based biology. This approach is rapidly superseding earlier approaches based on “positional cloning” and other methods. Functional genomics relies heavily on the various tools of bioinformatics to identify gene sequences of potential interest from the many molecular biology databases now available as well as from other sources. There is a continuing and significant need to identify and characterize further genes and other polynucleotides sequences and their related polypeptides, as targets for drug discovery.
Clearly, there exists a need for polynucleotides and polypeptides, such as the FabK embodiments of the invention, that have a present benefit of, among other things, being useful to screen compounds for antimicrobial activity. There is also a need for identification and characterization of such polynucleotides and polypeptides, and their antagonists and agonists in order to find ways to prevent, ameliorate or correct such infection, dysfunction and disease.
A FabK enzyme, involved in fatty acid biosynthesis, has been recently reported from a strain of Streptococcus pneumoniae (Heath, et al. Nature 406: 145 (2000)). The present invention provides a variant form of FabK, differing at Thr 318.
The specific activity of the known enzyme under the published conditions was 64+/−4 nmol min−1, too low to efficiently screen for compounds that modulate the activity of the enzyme, such as inhibitors (Heath, et al. Nature 406: 145 (2000)). The present invention solves this problem by providing a variant of the known FabK described as well as method for screening for FabK agonists and antagonists, wherein FabK activity is sufficient to perform efficient compound screening.
Moreover, the present invention provides a method of a diagnosing bacterial infection and bacterial genotyping using FabK polynucleotides and polypeptides.